Anti-citrulline antibodies are specific and predictive of rheumatoid arthritis (RA). Seropositivity against citrulline is clinically assayed using cyclic citrullinated peptide (CCP) ELISA. Despite being an excellent proxy assay for diagnosis, anti-CCP positivity does not reveal any information about the actual underlying antigens that elicited the immune response. Recent studies demonstrated the value of identifying autoantibodies to particular antigens in the elucidation of RA etiology. Furthermore, the identification of specific citrullinated antigens might help improve the assay performance of anti-CCP test. Unfortunately, only a few citrullinated antigens have been discovered in the past several decades. Developing a platform that will identify antibodies against citrullinated proteins will not only help understad the disease pathogenesis but also improve diagnosis. Traditional protein immuno-chemistry methods to identify citrullinated antigens suffer drawbacks such as low throughput, poor reproducibility, inadequate quantification and low resolution. Commercial protein arrays are expensive, lack equal representation of candidate antigens and are not compatible with post-translational modifications such as citrullination, which requires harsh conditions. We propose a cover capture protein microarray platform where proteins are expressed in silicon microwells and captured as naked protein by ligands on the cover slides. Our development will be based on our innovative NAPPA protein microarray platform to circumvent challenges associated with commercial protein arrays for spotting purified recombinant proteins. NAPPA involves printing full length cDNAs corresponding to proteins of interest on the microarray substrate and then transcription/translation in situ at the time of assay. Cover capture NAPPA will enable just-in-time proteins expression and citrullination on array post-translationally without the interference of NAPPA printing mixture components. Captured proteins will be presented on standard microscopic slides and the employment of high affinity tag/ligand will enable a variety of post-translational modifications on array including citrullination. Sero- reactivity against multiple citrullinated proteins can then be assessed in parallel. We will establish this platform to profile anti-citrullinated protein antibodies in RA at the proteome level and to map immuno-dominant epitopes of citrullinated autoantigens on array with the key advantages being high-throughput, multiplexed, quantitative, and low-cost so that enough samples can be assayed to draw statistical sound conclusion. Our goal is to develop a high-throughput platform to discover additional antigens, when citrullinated, can be recognized by antibodies in RA patients at the proteome level. We believe cover-capture NAPPA will enable this discovery and greatly benefit RA research to understand disease pathogenesis and develop more sensitive diagnostics enabling patient stratification based on autoantigens. Furthermore, what we propose to develop can be applied to studies in understanding the biology of other post-translational modifications. PUBLIC HEALTH RELEVANCE: A protein array platform for anti-citrulline antibodies in Rheumatoid Arthritis Project Narrative/Relevance Three million Americans suffer Rheumatoid Arthritis. One characterizing feature is the development of antibodies against citrulline modified self proteins. We will develop a platform that enables the screening of the entire repertoire of citrulline modified human proteins in parallel and the identification of RA specific antibodies. Th identification of disease specific antibodies will significantly accelerate RA research.